The enzymatic cleavage of double-stranded RNA is an essential step in the maturation and degradation of diverse eukaryotic and prokaryotic RNAs, and is a key event in RNA interference (RNAi) and related gene silencing and genome maintenance mechanisms. dsRNA cleavage is catalyzed by members of the ribonuclease III (RNase III) family of endoribonucleases, which are highly conserved in bacterial and eukaryotic cells. Eukaryotic RNase III orthologs include Dicer and Drosha, which carry out the maturation of microRNAs and other gene-regulatory RNAs. RNase III also is a component of RNA editosomes in trypanosome mitochondria. dsRNA processing by bacterial ribonucleases III regulates cellular and phage gene expression, plasmid replication, antibiotic production, and virulence factor expression. Bacterial RNase substrates are cleaved in a highly site-specific manner, which is required for proper half-life or optimal function of the RNAs. There also is evidence that bacterial RNase III can regulate gene expression by binding RNA in a non-catalytic mode. The long-term objective of this project is to characterize the RNA sequence and structural elements and RNA-protein interactions that confer specificity in RNase III substrate recognition and cleavage. Specific Aim 1 will employ in vitro selection, nucleotide analog interference mapping and site-directed mutagenesis to identify substrate functional groups essential for recognition. Specific Aim 2 will characterize protein functional group contributions to substrate specificity and binding energy. Site-directed mutagenesis and polypeptide segment exchange experiments will identify RNase III domains involved in specificity of binding. Specific Aim 3 will define the sequence and structural features of catalytic antideterminants in two specific RNAs. In vitro selection, site-directed mutagenesis, and RNA structure-probing will be used to determine the critical structural and functional features of an in vitro-selected, binding-competent RNA, and the lambda phage cIII mRNA 5'-leader. The mechanism of RNase III activation will be analyzed using an in vitro translation system. Along with Escherichia coli RNase III, this project will employ Aquifex aeolicus RNase III and Thermotoga maritima RNase III, for which structural data is available. These analyses will serve to define how specificity is achieved in dsRNA processing by RNase III, and ultimately determine the role of dsRNA processing in the cellular defense mechanisms, including RNAi and in specific disease states, including cancer and infectious diseases.